1. Field of the Invention
This invention is related in general to electrodes for electrophysiological testing of biological membranes. In particular, the invention concerns the manufacture of an electrode assembly suitable for massive parallel electrophysiological testing of cells, and a method for rapidly and reliably positioning the cells in each chamber for forming a high electrical-resistance patch clamp.
2. Description of the Related Art
In conventional voltage-clamping techniques used to conduct electrophysiological tests on a membrane, the electrical activity in the membrane is assessed by measuring current or voltage changes produced in response to exposure to various test stimuli. Typically, the membrane is pierced with two microelectrodes connected to an amplifier capable of recording current or voltage variations in response to stimuli such as voltage step changes, current injection, the application of compounds, or mechanical stimulation.
Similarly, using patch-clamping techniques, the membrane potential can be held constant while the current flowing through the membrane is measured to detect ion-channel activity that corresponds to changes in the membrane""s conductance. Instead of using sharp microelectrodes to puncture the membrane and penetrate the cell, like in traditional voltage clamping, patch clamping uses a micropipette with a heat-polished tip of about 1 to 5 xcexcm in diameter that is physically sealed to a xe2x80x9cpatchxe2x80x9d on the membrane. The same pipette is used continuously for both current passing and voltage recording.
For the most part, patch clamping is used either in a whole-cell or a single-channel mode of operation. In whole-cell patch clamping, the membrane at the tip of the pipette is ruptured to produce electrical continuity between the electrolyte in the pipette and the interior of the cell. Thus, total membrane current or voltage is measured. In single-channel patch clamping, the integrity of the membrane at the tip of the pipette is preserved. Accordingly, the recorded current is only the current flowing through the patch of the membrane enclosed by the tip of the pipette. Since this area is very small, there is a good chance that only one or a small number of ion channels may be in the membrane patch, and individual ion-channel currents may be recorded.
In both types of patch-clamp techniques, when the tip of the pipette is pressed against the cell membrane, the interior of the pipette is isolated from the extracellular solution by the seal that is formed between the tip of the pipette and the membrane. If the electrical resistance of the seal is very high (in the order of several hundred mega-ohm to one giga-ohm), no current can leak across the seal and good measurements are obtained. Thus, any leakage of current through the seal is undesirable and the creation of a high-resistance seal (in the order of giga-ohms) is crucial for good results.
New patch-clamp electrodes have recently been developed in the art based on a seal formed by a test cell""s membrane and an aperture in a nonconductive partition separating the extracellular carrier solution from an intracellular electrolyte. The plate configuration of the partition allows the manufacture of trays with multiple perfusion chambers for parallel testing of large numbers of cells. Accordingly, new insulating seal materials and patch-clamp seal geometries are being investigated in the art to improve the quality of the seals and the speed of testing in such parallel operations. Typically, these new systems comprise multiple perfusion chambers where the partition separates an upper (extracellular) compartment, where the test cells are suspended in an extracellular solution toward the aperture for forming a patch-clamp seal with it, from a bottom (intracellular) compartment containing an electrode and an electrolytic solution. For instance, Cytion""s International Application No. PCT/IB98/01150 describes a perforated partition with multiple holes to form a plurality of patch-clamp seals between intra and extracellular compartments. In International Application No. PCT/EP00/08895, a system is described where a pulsatile negative pressure is used to position the test cell on a perforated surface in a high-resistance seal arrangement. The suction is also used to penetrate the membrane of the cell and connect its interior to the ambient electrolytic solution. Similarly, International Application No. PCT/GB00/04887 discloses a system where a test cell is drawn to form a seal in an aperture in a well by suction through the well and through lateral channels that increase fluid flow in the desired direction. These channels are also used to remove the cell by reversing the direction of flow.
In addition to the ability to form a high-resistance seal, good electrophysiological testing requires that each cell be placed on the electrode aperture without excessive mixing (optimally with no mixing) of extracellular solution (contained in the upper extracellular compartment) with the intracellular electrolytic solution contained in the lower intracellular compartment. When such mixing occurs, the homogeneity of the intracellular solution is reduced and its function is adversely affected for proper recording of electrophysiological responses. Therefore, systems that apply suction to place the test cells on the electrode apertures are subject to erroneous measurements that detrimentally affect their performance. This invention provides a different approach to solve this problem.
An important objective of this invention is a method of positioning a cell or other biological membrane on an electrode aperture rapidly and with minimal mixing of extracellular and intracellular solutions to form a high-resistance seal.
Another object is a positioning procedure and apparatus that afford a high degree of control over the flow of the extracellular solution and the intracellular solution in the system.
Still another objective of the invention is a high-resistance electrode assembly suitable for the parallel testing of large numbers of biological membranes, such as animal cells, through successive exposures to multiple perfusion solutions in a continuous, high-throughput operation.
Another goal is an electrode assembly design that is suitable for implementation within an overall automated high-resistance patch-clamp and solution-delivery system.
Another objective is a modular method of manufacture of the electrode assembly, such that each component may be fabricated independently with known techniques and combined to implement the precise structural details required for the electrical and fluidic systems of electrophysiological perfusion chambers.
Yet another object is a system that can be implemented using conventional patch-clamp electronic hardware and software, modified only to the extent necessary to meet the design parameters of the electrode assembly of the invention.
A final objective is a system that can be implemented economically according to the above stated criteria.
Therefore, according to these and other objectives, the present invention consists of a perfusion-chamber structure that includes a chamber plate with an extracellular compartment, a partition plate with an electrode aperture, and a foundation plate with an intracellular compartment. A gap between the chamber plate and the partition plate produces a channel for applying suction that draws extracellular solution from the extracellular compartment and facilitates the movement and positioning of a test cell over the electrode aperture. According to a very important aspect of the invention, the suction portion of the positioning procedure for the test cell is accompanied by a slight positive pressure applied to the intracellular solution in the intracellular compartment of the chamber to cause upward flow of the intracellular solution through the electrode aperture. As a result of the concurrent suction and pressure flows produced in the vicinity of the electrode aperture, the test cell is separated from impurities that may be present in the extracellular solution, thereby permitting a clean contact with the electrode aperture and correspondingly the formation of a high-resistance seal. Because of the upward flow of intracellular solution through the electrode aperture, the intracellular compartment of the chamber is not contaminated with extracellular solution, thereby avoiding the detrimental effects experienced when only suction is used to draw the test cell to the electrode aperture. When the cell is positioned over the electrode aperture, the positive pressure on the intracellular fluid is reversed to suction and the cell is seated thereby to form the seal.
According to another aspect of the invention, an electrode assembly for parallel electrophysiological recording of large numbers of cells comprises multiple plate components suitable for combination and connection with conventional electronic and fluid transfer systems. In the preferred embodiment, the assembly includes a top disposable section and a bottom permanent section. The top section comprises a large number of perfusion chambers for parallel testing, each chamber having an upper funnel-like compartment and a lower tubular channel separated by an electrode aperture adapted to receive a cell from an extracellular carrier solution. The bottom section comprises mating electrical couplers and tubular flow ports aligned with corresponding metallic leads and channels in the upper section for connection to computer-controlled electronic and fluid-flow hardware. The two sections are combined for operation in a simple, clamped arrangement that ensures hermetic and insulated connection of the fluidic and electrical systems, respectively. The electrode assembly is made with various plate components that may be produced independently and combined to form the top and bottom sections. These plates may be manufactured with precise definition utilizing etching and other techniques known in the art of electronic-component fabrication.
Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose but one of the various ways in which the invention may be practiced.